Flavobacterium columnare • Columnaris disease – Described by Davis in 1922 – Bacillus columnare, Flexibacter columnaris, Cytophaga columnaris, Flavobacterium columnare… – Columnaris disease affects wild, farm- raised and ornamental fish species • Main disease for channel ca@ish 1 Columnaris disease in ca@ish • Mortality rates 30 – 50-60% in food fish 20 – Up to 90% in fingerlings • Epizooology 10 Percentage columnaris per month – Early spring & late summer J F M A M J J A S O N D – Acute and chronic manifestaIons – Environmental stressors GeneIc heterogeneity • Three genomovar described within the species – Genomovar I • Europe • USA – Genomovar II • USA • Japan – Genomovar III • Japan • USA 2 Virulence • Genomovar II is more virulent than genomovar I in channel ca@ish Strain Genomovar Dose Mean CumulaIve % mortality LSU II 7.0 x 105 100.0 (±0) 530 II 7.2 x 105 100.0 (±0) 515 II 7.7 x 105 96.2 (±0) 02-26 II 6.9 x 105 92.3 (±0) ARS-1 I 7.3x 105 46.2 (±0) HS I 7.5 x 105 3.9(±0) 463 I 7.7 x 105 0.0 (±0) Control NA NA 0.0 (±0) Shoemaker, C.A., Olivares-Fuster, O., Arias, C.R. & Klesius, P.H. (2008) Veterinary Microbiology 127 353-359. Specific host-species associaIon • In wild fish populaIons genomovar II has been found almost exclusively associated to ca@ish Threadfin shad Freshwater drum Channel catfish II Blue catfish I Olivares-Fuster, O., Baker, J.L., Terhune, J.S., Shoemaker, C.A., Klesius, P.H. & Arias, C.R. (2007). SystemaIc and Applied Microbiology 30, 624-633. 3 Early stages of host-pathogen interacIon • Chemotaxis towards ca@ish mucus – Genomovar II has a higher chemotaxis response towards ca@ish mucus than genomovar I Klesius, P.H., Shoemaker, C.A. & Evans, J.J. (2009) FEMS Microbiology Leeers 288, 216-220. • Adhesion to gill Issue plays main role in colonizaIon and invasion – Contradictory studies on correlaIon between virulence and adhesion in F. columnare Decostere, A., Haesebrouck, F., Van Driessche, E., Charlier, G. & Ducatelle, R. (1999) Journal of Fish Diseases 22, 465-474. Kuneu, H.M.T., Suomalainen, L.-R., Jokinen, R.I. & Valtonen, E.T. (2009) Microbial Pathogenesis 46, 21-27. Objecve • To compare adhesion of Flavobacterium columnare genomovar I and II to channel ca@ish and zebrafish 4 Material and Methods • StaIc aquaria (35 L) – Each tank is an independent unit – 15 or 35 fish/tank (ca@ish/zebrafish) • Bath immersion – 30 min with ~106 CFU/ml – No stressor • Treatments (4 tanks/treatment) – ARS-1 genomovar I – BGFS 27 genomovar II – Control – Terminated at 24 h post-challenge Materials and Methods, cont. • Fish – Ca@ish: experiment 1 and 3 – Zebrafish: experiment 2 and 4 • Samples – Gill clips at 0.5, 6, 12 and 24 h post-challenge (+ control pre-challenge) – Skin ~0.5 cm2 (Experiment 1 and 2) – 3 fish pooled/treatment X 3 – F. columnare CFU/g (selecve medium) – Scanning electron microscopy (SEM) 5 Results • Experiment 1: Ca@ish 1.00E+07 1.00E+06 * 1.00E+05 * * 1.00E+04 CFU/g ARS-1 1.00E+03 BGFS-27 1.00E+02 1.00E+01 1.00E+00 0.5 1 6 12 24 Post-challenge time (hour) Results Mortality • Experiment 3: Ca@ish 1.00E+07 * 1.00E+06 * 1.00E+05 * * * 1.00E+04 ARS-1 CFU/g 1.00E+03 BGFS-27 1.00E+02 1.00E+01 1.00E+00 0.5 1 6 12 24 Post-challenge time (hour) 6 Results • Experiment 2: Zebrafish 1.00E+08 * 1.00E+07 * 1.00E+06 1.00E+05 CFU/g 1.00E+04 * * ARS-1 * BGFS-27 1.00E+03 1.00E+02 1.00E+01 1.00E+00 0.5 1 6 12 24 Post-challenge time (hour) Results • Experiment 4: Zebrafish * 1.00E+07 * * 1.00E+06 1.00E+05 1.00E+04 * ARS-1 CFU/g 1.00E+03 * BGFS-27 1.00E+02 1.00E+01 1.00E+00 0.5 1 6 12 24 Post-challenge time (hour) 7 Results • Genomovar I: ca@ish vs zebrafish 1.00E+07 * * No columnaris signs 1.00E+06 1.00E+05 1.00E+04 * * ARS-C CFU/g 1.00E+03 ARS-Z * 1.00E+02 1.00E+01 1.00E+00 0.5 1 6 12 24 Post-challenge time (hour) Results Columnaris signs • Genomovar II: ca@ish vs zebrafish and mortality observed in both 1.00E+08 * species 1.00E+07 * * 1.00E+06 * 1.00E+05 1.00E+04 BGfS-27-C CFU/g BGFS-27-Z 1.00E+03 1.00E+02 1.00E+01 1.00E+00 0.5 1 6 12 24 Post-challenge time (hour) 8 Results • No difference between ARS-1 and BGFS-27 observed by SEM (@ 0.5 h post-challenge) Conclusions • Marked difference in adhesion dynamics between genomovars I and II – Both genomovars can adhere to ca@ish gills at high levels (>104 CFU/g) within 1 h post-challenge – Genomovar II adheres to zebrafish gill Issue at significantly higher levels than genomovar I – Genomovar II persists longer (24 h) at higher numbers (>103 CFU/g) in both species 9 Conclusions cont. • Adhesion of F. columnare to gill Issue is insufficient to cause columnaris disease • Persistence in gill Issue is beeer correlated with virulence of F. columnare strains than aeachment – CriIcal to follow the dynamics of adhesion: analyze more than one Ime point Acknowledgments • Funding sources • Dr. Newton, School of Veterinary Sciences, Auburn University • Dr. Miller, Director of the Advance Microscopy & Imaging Lab, Auburn University 10 .